Oligonucleotide compositions and their use to induce differentiation of cells

ABSTRACT

The present invention provides compositions comprising a 3′-OH, 5′-OH, chemically unmodified, synthetic phosphodiester nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and a pharmaceutically acceptable carrier, wherein the compositions are useful to induce differentiation of cells or to stimulate differentiation or production of pluripotent cells. The present invention provides methods of using these compositions to induce differentiation of pluripotent cells, including bone marrow derived cells, and to treat disease associated with insufficient differentiation of cells in animals and humans, including but not limited to leukemia, lymphoma, non-malignant blood disorders such as hemoglobinopathies, sickle cell disease, myelodysplastic syndrome, pancytopenia, anemia, thrombocytopenia and leukopenia.

PRIOR RELATED APPLICATIONS

The present application claims priority to U.S. provisional patentapplication Ser. No. 60/286,158 filed Apr. 24, 2001. This application isa divisional of U.S. patent application Ser. No. 10/127,645 filed Apr.22, 2002, now allowed, the contents of which are incorporated herein byreference.

SEQUENCE LISTING

The content of the sequence listing information is identical to thepaper sequence listing provided in U.S. patent application Ser. No.10/127,645 filed Apr. 22, 2002, and includes no new matter.

FIELD OF THE INVENTION

The present invention provides compositions comprising specificoligonucleotides combined with a pharmaceutically acceptable carrier,wherein the compositions are useful to induce differentiation of cells,including pluripotent cells, leukemic cells, lymphoma cells and bonemarrow-derived cells, and to treat diseases such as leukemia, lymphomaand disorders associated with insufficient differentiation of cells.

BACKGROUND OF THE INVENTION

Numerous diseases and conditions in animals and humans are associatedwith insufficient differentiation of cells or with an insufficiency ofcells. Many of these cells are derived from bone marrow. Such diseasesand conditions include but are not limited to leukemia, lymphoma, andnon-malignant blood disorders such as hemoglobinopathies, sickle celldisease, myelodysplastic syndrome and insufficient production of bonemarrow derived cells following therapies such as radiation andchemotherapy.

Differentiation therapy of leukemia cells in diseases such as acutepromyelocytic leukemia (APL), acute myeloid leukemia (AML), chronicpromyelocytic leukemia (CPL) and chronic myeloid leukemia (CML), hasprovided an alternative strategy for the treatment of leukemia. Indifferentiation therapy, immature leukemia cells are induced bydifferent chemical compounds to attain a mature phenotype resulting inarrest of their growth.

A number of differentiation compounds and also radiation have beenreported to induce the differentiation of leukemia cells. Hemin, butyricacid, 5-azacytidine, cytosine arabinoside, hydroxyurea, guanosine,guanine, retinoic acid, trimidox, gamma-irradiation, mithramycin andchromomycin have been reported to induce differentiation of leukemiacells (Rutherford et al., Nature 280:164, 1979; Gambari et al., Biochem.Biophys. Acta, 886:203, 1986; Bianchi et al., Cancer Res. 46:6327, 1986;Adunyah et al., Biochem. Biophys. Acta, 1263:123, 1995; Osti et al.,Haematologica 82:395, 1997; Cortesi et al., Eur. J. Haematol. 61:295,1998; Iyamu et al., Biochem. Biophys. Res. Com. 247:759, 1998; Schwenkeet al., Leuk. Res. 19:955, 1995; and Bianchi et al., Br. J. Haematol.104:258, 1999).

Synthetic oligonucleotides are polyanionic sequences that areinternalized in cells (Vlassov et al. Biochim. Biophys. Acta 1197:95,1994). Synthetic oligonucleotides are reported to bind selectively tonucleic acids (Wagner, R. Nature: 372:333, 1994), to specific cellularproteins (Bates et al. J. Biol. Chem. 274:26369, 1999) and to specificnuclear proteins (Scaggiante et al. Eur. J. Biochem. 252:207, 1998), andto inhibit proliferation of cancer cells. Synthetic oligonucleotideshave not been reported to possess differentiating activity on acuteand/or chronic pro-myelocytic cells and/or myeloid leukemia cells.Synthetic phosphorothioate oligonucleotides having a CpG motif(5′purine-purine-cytosine (C)-guanine (G)-pyrimidine-pyrimidine3′) havebeen shown to induce the proliferation of B-cell chronic lymphocyticleukemia (Decker et al., Blood 95:999, 2000). Synthetic 27 basesequences containing G and variable amounts of thymine (T), hereinafteroligonucleotide GTn, wherein n is ≧1 or ≦7 Ts (Scaggiante et al., Eur.J. Biochem. 252:207, 1998), and wherein the number of bases is >20(Morassutti et al., Nucleosides and Nucleotides 18:1711, 1999), havebeen reported to inhibit growth of leukemia cells by sequence specificbinding to a 45 kDa nuclear protein. In contrast, GTn sequences, whereinthe total number of bases is less than 15, are reported to be inactiveagainst these cells (Morassutti et al. Nucleosides and Nucleotides18:1711, 1999). Chimeric methylphosphonodiester/phosphodiesteroligonucleotides of sequence type SEQ ID NO:5 CGNNN (N=A, C, G or T),introduced into the cytoplasm of cells by 10 minutes of reversiblepermeabilization with streptolysin O, induce apoptosis of human T cellleukemia cells. Nevertheless, the CGNNN oligonucleotides are reported tobe inactive against three CML cell lines (K562, LAMA84 and KYO1),showing no significant effect on the growth and survival of these cells(Tidd et al., Nucleic Acid Res. 28:2242, 2000).

Depletion of bone marrow derived cells is observed in severalconditions, including depletion following radiation therapy orchemotherapy. Insufficient production of cells destined to becomeerythrocytes or granulocytes is associated with numerous problems,including but not limited to, reduced delivery of oxygen to cells,decreased immune function, and clotting abnormalities. Varioustherapies, including expensive chemotherapies, are often required tostimulate production of red and white cells.

Most prior art differentiating therapies have proven to be less thanadequate for clinical applications. Many of these therapies areinefficient or toxic, have significant adverse effects and aredebilitating for the recipient. Therefore, there is a continuing needfor novel compositions and methods that induce differentiation of cellssuch as myeloid-derived leukemia cells. What is also needed are newtherapeutic compositions and methods that stimulate production anddifferentiation of pluripotent cells such as bone-marrow derived cells.Also needed are new therapeutic compositions that induce differentiationof cells. What is also needed are compositions and methods that may beused to treat diseases and conditions characterized by insufficientdifferentiation of cells or insufficient production of marrow derivedcells.

SUMMARY OF THE INVENTION

The present invention fulfills these needs by providing a methodcomprising administration of a composition comprising a 3′-OH, 5′-OH,chemically unmodified, synthetic phosphodiester oligonucleotide sequence(hereinafter sequence) selected from the group consisting of SEQ ID NO:1 (5′GTG3′), SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3 (5′GGGAGG3′) andSEQ ID NO: 4 (5′CCACCC3′) and a pharmaceutically acceptable carrier,wherein the composition induces differentiation of cells. The presentinvention provides a method to treat diseases associated with growth ofcells that are not differentiated to a mature phenotype. Terminaldifferentiation of cells may include one or more responses selected fromthe group consisting of induction of erythrocyte-like phenotype,monocyte-like phenotype, megakaryocyte-like phenotype, inhibition ofproliferation of leukemia cells and induction of hemoglobin synthesis.

The compositions of the present invention may be used to treat diseasesrelated to insufficient differentiation of cells. Such diseases includebut are not limited to leukemia, lymphoma, and non-malignant blooddisorders such as hemoglobinopathies, sickle cell disease ormyelodysplastic syndrome. The compositions of the present invention arebelieved to be useful for treatment of pancytopenia, anemia,thrombocytopenia and leukopenia. Other conditions that may be treatedwith the compositions of the present invention include lymphoma andnonmalignant blood disorders, including but not limited tohemoglobinopathies, sickle cell disease and myelodysplastic syndromes.In a preferred embodiment, the compositions of the present invention areadministered to an animal or a human with leukemia in an amounteffective to treat the leukemia.

The compositions of the present invention may also be administered to ananimal or human together with other therapies as a combination therapy.These therapies may include administration of therapeutic compounds orradiation therapy. The compositions of the present invention may beadministered before, after, or concomitantly with the other therapy.Such combination therapy may augment the net therapeutic effect on theanimal or human. The compositions of the present invention may beadministered alone, or in combination with other therapeutic modalitiesincluding, but not limited to, chemotherapeutic agents, differentiatingagents, immunotherapeutic agents, antimicrobial agents, antiviral agentsor in combination with radiation therapy.

The compositions of the present invention may be administered to arecipient to stimulate production of cells after other therapiesadministered to the recipient have depleted such cells. One non-limitingexample involves depletion of cells derived from bone marrow followingradiation therapy or chemotherapy. The compositions of the presentinvention may also be administered to a recipient to stimulateproduction and differentiation of other cells such as pluripotent stemcells, myeloid stem cells, lymphoid stem cells, progenitor cells, immunecell precursors, and/or other cells derived from these pluripotent stemcells, myeloid stem cells, lymphoid stem cells, progenitor cells, andimmune cell precursors. The compositions of the present invention mayalso be administered to a recipient to stimulate production anddifferentiation of cells from numerous sources, including but notlimited to, bone marrow, liver, spleen, lymph nodes, thymus and cordblood.

The compositions of the present invention may also be administered invitro to affect differentiation of cells such as pluripotent stem cells,myeloid stem cells, lymphoid stem cells, progenitor cells, immune cellprecursors, and/or other cells derived from these pluripotent stemcells, myeloid stem cells, lymphoid stem cells, progenitor cells, andimmune cell precursors.

The unexpected and surprising ability of the composition of the presentinvention to induce differentiation of bone-marrow derived cells,including leukemia cells, addresses a long-felt, unfulfilled need in themedical arts and provides an important benefit for animals and humans.

Accordingly, it is an object of the present invention to provide amethod comprising administration of a composition comprising a 3′-OH,5′-OH, chemically unmodified, synthetic phosphodiester oligonucleotidesequence (hereinafter sequence) selected from the group consisting ofSEQ ID NO: 1 (5′GTG3′), SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3(5′GGGAGG3′) and SEQ ID NO: 4 (5′CCACCC3′) and a pharmaceuticallyacceptable carrier to treat disease in animals and humans, wherein thedisease is characterized by insufficient differentiation of cells.

Another object of the present invention is to provide a methodcomprising administration of a composition comprising a 3′-OH, 5′-OH,chemically unmodified, synthetic phosphodiester oligonucleotide sequence(hereinafter sequence) selected from the group consisting of SEQ ID NO:1 (5′GTG3′), SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3 (5′GGGAGG3′) andSEQ ID NO: 4 (5′CCACCC3′) and a pharmaceutically acceptable carrier toinduce progenitor cell maturation and differentiation in animals andhumans.

Another object of the present invention is to provide a composition andmethod to treat leukemia.

Yet another object of the present invention is to provide a method thatinhibits proliferation of leukemic cells and induces differentiation ofleukemic cells.

Another object of the present invention is to provide a method to treatlymphoma.

Yet another object of the present invention is to provide a method totreat non-malignant blood disorders.

Still another object of the present invention is to provide a method totreat hemoglobinopathies.

A further object of the present invention is to provide a method totreat sickle cell disease.

Yet another object of the present invention is to provide a method totreat myelodysplastic syndrome.

Another object of the present invention is to provide a method to treatpancytopenia.

Yet another object of the present invention is to provide a method totreat anemia.

A further object of the present invention is to provide a method totreat thrombocytopenia.

Another object of the present invention is to provide a method to treatleukopenia.

Still another object of the present invention is to provide a method toinduce progenitor cell maturation and differentiation.

Yet another object of the present invention is to provide a method toinduce maturation and differentiation of cells including but not limitedto pluripotent stem cells, myeloid stem cells, lymphoid stem cells,progenitor cells, immune cell precursors, and/or other cells derivedfrom these pluripotent stem cells, myeloid stem cells, lymphoid stemcells, progenitor cells, and immune cell precursors.

Still another object of the present invention is to provide a method toinduce bone marrow-derived progenitor cell maturation.

Another object of the present invention is to provide a method thatincreases the number of bone marrow derived-cells following treatmentwith therapeutic agents.

Yet another object of the present invention is to provide a method thatincreases the number of bone marrow derived-cells following treatmentwith chemotherapeutic agents.

Another object of the present invention is to provide a method thatrestores the number of bone marrow derived-cells following treatmentwith radiotherapy.

Still another object of the present invention is to provide a methodthat restores the number of bone marrow derived-cells followingtreatment with immunosuppressive agents.

Still another object of the present invention is to provide acomposition that is minimally toxic to the recipient.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments included herein.

The present invention comprises a method comprising administration of acomposition comprising a 3′-OH, 5′-OH, chemically unmodified, syntheticphosphodiester oligonucleotide sequences (hereinafter sequence) selectedfrom the group consisting of SEQ ID NOs: 1, 2, 3, or 4, and apharmaceutically acceptable carrier, to an animal or a human in anamount effective to induce differentiation of cells. The compositions ofthe present invention may be used to treat disease related toinsufficient differentiation of cells. Such diseases include but are notlimited to leukemia, lymphoma, and non-malignant blood disorders such ashemoglobinopathies, sickle cell disease and myelodysplastic syndrome.The compositions of the present invention are believed to be useful fortreatment of pancytopenia, anemia, thrombocytopenia and leukopenia.Other conditions that may be treated with the compositions of thepresent invention include lymphoma and nonmalignant blood disorders,including but not limited to hemoglobinopathies, sickle cell disease andmyelodysplastic syndromes. The unexpected and surprising ability ofthese compositions to induce differentiation and to inhibitproliferation of leukemia cells addresses a long felt unfulfilled needin the medical arts and provides an important benefit for animals andhumans.

The present invention also comprises a method comprising administrationof a composition comprising a 3′-OH, 5′-OH, chemically unmodified,synthetic phosphodiester oligonucleotide sequence (hereinafter sequence)selected from the group consisting of SEQ ID NO: 1 (5′GTG3′), SEQ ID NO:2 (5′GGGTGG3′), SEQ ID NO: 3 (5′GGGAGG3′) and SEQ ID NO: 4 (5′CCACCC3′)and a pharmaceutically acceptable carrier, to induce maturation ofprogenitor cells in animals and humans. Such cells include but are notlimited to pluripotent stem cells, myeloid stem cells, lymphoid stemcells, immune cell precursors, and/or other cells derived from thesepluripotent stem cells, myeloid stem cells, lymphoid stem cells andimmune cell precursors. The compositions of the present invention mayalso be administered to an animal or human to stimulate production anddifferentiation of cells from numerous sources, including but notlimited to, bone marrow, liver, spleen, lymph nodes, thymus and cordblood.

As used herein, the word “sequence” refers a sequence comprising a3′-OH, 5′-OH chemically unmodified, synthetic phosphodiester nucleotidesequence selected from the group consisting of SEQ ID NO: 1 (5′GTG3′),SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3 (5′GGGAGG3′) and SEQ ID NO: 4(5′CCACCC3′).

The word “response”, as used herein refers to one or more of thefollowing non-limiting examples of responses: induced differentiation ofpluripotent stem cells, myeloid stem cells, lymphoid stem cells, immunecell precursors, and/or other cells derived from these pluripotent stemcells, myeloid stem cells, lymphoid stem cells and immune cellprecursors; induced differentiation of erythrocyte-like cells,monocyte-like cells or megakaryocyte-like cells; inhibition of cellularproliferation due to the induction of terminal differentiation;induction of hemoglobin synthesis; and stimulation of hemoglobinsynthesis.

As used herein, the phrase “effective in responsive cells” refers to theability of the compositions of the present invention to inducedifferentiation and/or inhibition of proliferation and/or synthesis ofhemoglobin.

As used herein, the phrases “therapeutic treatment”, “effective amount”and “amount effective to” refer to an amount of a sequence effective toinduce differentiation of cells, to inhibit proliferation of cells or tostimulate production of pluripotent cells such as bone marrow-derivedcells.

The word “disease”, as used herein, relates to a condition whereinbodily health is impaired. As used herein, the phrase “chemotherapeutic”is any agent approved by a regulatory agency of a country or a stategovernment or listed in the U.S.

Pharmacopoeia or other generally recognized pharmacopoeia for use totreat cancer in an animal or human. As used herein, the phrase“chemotherapeutic” includes immunosuppressive agents. Administration ofan effective amount of the composition of the present invention to ananimal or a human is a therapeutic treatment that prevents, treats oreliminates a disease including, but not limited to, leukemia,pancytopenia, anemia, thrombocytopenia, leukopenia, lymphoma, andnon-malignant blood disorders such as hemoglobinopathies, sickle celldisease or myelodysplastic syndrome. Types of leukemia include, but arenot limited to APL, AML, CPL and CML. Administration of an effectiveamount of the composition of the present invention to an animal or ahuman is also a therapeutic treatment that stimulates production ofprogenitor cells, including but not limited to pluripotent stem cells,myeloid stem cells, lymphoid stem cells, immune cell precursors, and/orother cells derived from these pluripotent stem cells, myeloid stemcells, lymphoid stem cells and immune cell precursors. In a preferredembodiment, the present invention provides a method to stimulateproduction and differentiation of marrow derived cells. The compositionsof the present invention may also be administered to an animal or humanto stimulate production and differentiation of cells from numeroussources, including but not limited to, bone marrow, liver, spleen, lymphnodes, thymus and cord blood.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable vehicle” are used herein to mean, without limitation, anyliquid, solid or semi-solid, including, but not limited to, water orsaline, a gel, cream, salve, solvent, diluent, fluid ointment base,ointment, paste, implant, liposome, micelle, giant micelle, and thelike, which is suitable for use in contact with living animal or humantissue without causing adverse physiological responses, and which doesnot interact with the other components of the composition in adeleterious manner. Other pharmaceutically acceptable carriers orvehicles known to one of skill in the art may be employed to makecompositions for delivering the oligonucleotide sequences of the presentinvention.

The oligonucleotide sequences of the present invention may be combinedwith pharmaceutically acceptable carriers and administered ascompositions in vitro or in vivo. Forms of administration include, butare not limited to, injections, solutions, creams, gels, implants,pumps, ointments, emulsions, suspensions, microspheres, particles,microparticles, nanoparticles, liposomes, pastes, patches, tablets,transdermal delivery devices, sprays, aerosols, or other means familiarto one of ordinary skill in the art. Such pharmaceutically acceptablecarriers are commonly known to one of ordinary skill in the art.Pharmaceutical formulations of the present invention can be prepared byprocedures known in the art using well known and readily availableingredients. For example, the compounds can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers that are suitable for such formulations include thefollowing: fillers and extenders (e.g., starch, sugars, mannitol, andsilicic derivatives); binding agents (e.g., carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone); moisturizing agents (e.g., glycerol);disintegrating agents (e.g., calcium carbonate and sodium bicarbonate);agents for retarding dissolution (e.g., paraffin); resorptionaccelerators (e.g., quaternary ammonium compounds); surface activeagents (e.g., cetyl alcohol, glycerol monostearate); adsorptive carriers(e.g., kaolin and bentonite); emulsifiers; preservatives; sweeteners;stabilizers; coloring agents; perfuming agents; flavoring agents;lubricants (e.g., talc, calcium and magnesium stearate); solid polyethylglycols; and mixtures thereof.

The formulations can be so constituted that they release the activeingredient only or preferably in a particular location, possibly over aperiod of time. Such combinations provide yet a further mechanism forcontrolling release kinetics. The coatings, envelopes, and protectivematrices may be made, for example, from polymeric substances or waxes.

Compositions comprising one or more sequences and a pharmaceuticallyacceptable carrier are prepared by uniformly and intimately bringinginto association the sequence and the pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers include liquid carriers,solid carriers or both. Liquid carriers are aqueous carriers,non-aqueous carriers or both, and include, but are not limited to,aqueous suspensions, oil emulsions, water-in-oil emulsions,water-in-oil-in-water emulsions, site-specific emulsions, long-residenceemulsions, sticky-emulsions, microemulsions and nanoemulsions. Solidcarriers are biological carriers, chemical carriers or both and include,but are not limited to, viral vector systems, particles, microparticles,nanoparticles, microspheres, nanospheres, minipumps, bacterial cell wallextracts and biodegradable or non-biodegradable natural or syntheticpolymers that allow for sustained release of the oligonucleotidecompositions. Emulsions, minipumps and polymers can be implanted in thevicinity of where delivery is required (Brem et al. J. Neurosurg. 74:441, 1991). Methods used to complex an oligonucleotide sequence(s) to asolid carrier include, but are not limited to, direct adsorption to thesurface of the solid carrier, covalent coupling to the surface of thesolid carrier, either directly or via a linking moiety, and covalentcoupling to the polymer used to make the solid carrier. Optionally, asequence(s) can be stabilized by the addition of non-ionic or ionicpolymers such as polyoxyethylenesorbitan monooleates (TWEENs) orhyaluronic acid.

Preferred aqueous carriers include, but are not limited to, water,saline and pharmaceutically acceptable buffers. Preferred non-aqueouscarriers include, but are not limited to, a mineral oil or a neutral oilincluding, but not limited to, a diglyceride, a triglyceride, aphospholipid, a lipid, an oil and mixtures thereof, wherein the oilcontains an appropriate mix of polyunsaturated and saturated fattyacids. Examples include, but are not limited to, soybean oil, canolaoil, palm oil, olive oil and myglyol, wherein the fatty acids can besaturated or unsaturated. Optionally, excipients may be includedregardless of the pharmaceutically acceptable carrier used to presentthe oligonucleotide compositions to cells. These excipients include, butare not limited to, anti-oxidants, buffers, and bacteriostats, and mayinclude suspending agents and thickening agents.

One or more sequences may be administered alone, or in combination withother therapeutic modalities including, but not limited to,chemotherapeutic agents, differentiating agents, immunotherapeuticagents, antimicrobial agents, antiviral agents or in combination withradiation therapy. Differentiating agents include, but are not limitedto, hemin, butyric acid, 5-azacytidine, cytosine arabinoside,hydroxyurea, guanosine, guanine, retinoic acid, trimidox,gamma-irradiation, mithramycin and chromomycin. Chemotherapeutic agentsinclude, but are not limited to, anti-metabolites, DNA damaging,microtubule destabilizing, microtubule stabilizing, actindepolymerizing, growth inhibiting, topoisomerase inhibiting, HMG-CoAinhibiting, purine inhibiting, pyrimidine inhibiting, metalloproteinaseinhibiting, CDK inhibiting, angiogenesis inhibiting, differentiationenhancing and immunotherapeutic agents. Dosages and methods ofadministration of these other therapeutic modalities are known to one ofordinary skill in the art.

Methods of in vivo administration of the compositions of the presentinvention, or of formulations comprising such compositions and othermaterials such as carriers of the present invention that areparticularly suitable for various forms include, but are not limited tothe following types of administration, oral (e.g. buccal or sublingual),anal, rectal, as a suppository, topical, parenteral, aerosol,inhalation, intrathecal, intraperitoneal, intravenous, intraarterial,transdermal, intradermal, subdermal, intramuscular, intrauterine,vaginal, into a body cavity, surgical administration at the location ofa tumor or internal injury, directly into tumors, into the lumen orparenchyma of an organ, and into bone marrow. Techniques useful in thevarious forms of administrations mentioned above include but are notlimited to, topical application, ingestion, surgical administration,injections, sprays, transdermal delivery devices, osmotic pumps,electrodepositing directly on a desired site, or other means familiar toone of ordinary skill in the art. Sites of application can be external,such as on the epidermis, or internal, for example a gastric ulcer, asurgical field, or elsewhere.

The compositions of the present invention can be applied in the form ofcreams, gels, solutions, suspensions, liposomes, particles, or othermeans known to one of skill in the art of formulation and delivery ofthe compositions. Ultrafine particle sizes can be used for inhalationdelivery of therapeutics. Some examples of appropriate formulations forsubcutaneous administration include but are not limited to implants,depot, needles, capsules, and osmotic pumps. Some examples ofappropriate formulations for vaginal administration include but are notlimited to creams and rings. Some examples of appropriate formulationsfor oral administration include but are not limited to: pills, liquids,syrups, and suspensions. Some examples of appropriate formulations fortransdermal administration include but are not limited to gels, creams,pastes, patches, sprays, and gels. Some examples of appropriate deliverymechanisms for subcutaneous administration include but are not limitedto implants, depots, needles, capsules, and osmotic pumps. Formulationssuitable for parenteral administration include but are not limited toaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffets, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletscommonly used by one of ordinary skill in the art.

Embodiments in which the compositions of the invention are combinedwith, for example, one or more pharmaceutically acceptable carriers orexcipients may conveniently be presented in unit dosage form and may beprepared by conventional pharmaceutical techniques. Such techniquesinclude the step of bringing into association the compositionscontaining the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers. Preferred unit dosage formulations are those containing a doseor unit, or an appropriate fraction thereof, of the administeredingredient. It should be understood that in addition to the ingredientsparticularly mentioned above, formulations comprising the compositionsof the present invention may include other agents commonly used by oneof ordinary skill in the art.

The volume of administration will vary depending on the route ofadministration. Such volumes are known to one of ordinary skill in theart of administering compositions to animals or humans. Depending on theroute of administration, the volume per dose is preferably about 0.001to 100 ml per dose, more preferably about 0.01 to 50 ml per dose andmost preferably about 0.1 to 30 ml per dose. For example, intramuscularinjections may range in volume from about 0.1 ml to 1.0 ml. Theoligonucleotide compositions administered alone, or together with othertherapeutic agent(s), can be administered in a single dose treatment, inmultiple dose treatments, or continuously infused on a schedule and overa period of time appropriate to the disease being treated, the conditionof the recipient and the route of administration. Moreover, the othertherapeutic agent can be administered before, at the same time as, orafter administration of the oligonucleotide compositions.

Preferably, the amount of oligonucleotide composition administered perdose is from about 0.0001 to 100 mg/kg, more preferably from about 0.001to 10 mg/kg and most preferably from about 0.01 to 5 mg/kg. In apreferred embodiment, the oligonucleotide compositions in combinationwith a chemotherapeutic agent is administered to an animal or humanhaving leukemia in an amount effective to add to, synergize with orpotentiate the anti-neoplastic effect of the chemotherapeutic agent.Preferably, the amount of therapeutic agent administered per dose isfrom about 0.001 to 1000 mg/kg, more preferably from about 0.01 to 500mg/kg and most preferably from about 0.1 to 100 mg/kg. The particularsequence and the particular therapeutic agent administered, the amountper dose, the dose schedule and the route of administration should bedecided by the practitioner using methods known to those skilled in theart and will depend on the type of disease, the severity of the disease,the location of the disease and other clinical factors such as the size,weight and physical condition of the recipient. In addition, in vitroassays may optionally be employed to help identify optimal ranges forsequence and for sequence plus therapeutic agent administration.

The compositions of the present invention may also be administered invitro to affect differentiation of cells such as pluripotent stem cells,myeloid stem cells, lymphoid stem cells, progenitor cells, immune cellprecursors, and/or other cells derived from these pluripotent stemcells, myeloid stem cells, lymphoid stem cells, progenitor cells, andimmune cell precursors.

The present invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention.

EXAMPLE 1

Preparation of Sequences

Phosphodiester nucleotide sequences (SEQ ID NOs: 1, 2, 3 and 4) wereprepared by Sigma-Genosys (Woodlands, Tex.) using Abacus SegmentedSynthesis Technology. Unless stated otherwise, the sequences weredispersed in autoclaved deionized water or in a pharmaceuticallyacceptable buffer such as, but not limited to, saline immediately priorto use.

EXAMPLE 2

Cells

The K562 cell line derived from the leukemic cells of a CML patient inblastic crisis is used as the standard model for determining, in vitro,the therapeutic potential of new differentiating compounds (Rutherfordet al., Nature, 280:164, 1979; Drexler et al. DSMZ Catalogue of Humanand Animal Cell Lines, 6^(th) ed., Braunschweig, Germany: DSMZ, 1997).K562 cells were obtained from the American Type Culture Collection(ATCC, Rockville, Md.) and were cultured in the medium recommended bythe ATCC.

EXAMPLE 3

Hemoglobin Synthesis by K562 Cells Cultured with SEQ ID NO. 1, SEQ IDNO. 2, SEQ ID NO: 3 and SEQ ID NO: 4.

K562 cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-wellflat-bottomed tissue culture plates for 72 hours with 100 μg of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. For hemoglobindetermination, the cells were washed twice by centrifugation inphosphate-buffered saline (PBS), stained with 0.2% benzidine(Sigma-Aldrich Canada, Oakville, Ontario) in 0.5 M acetic acid activatedwith 10% H₂O₂ (Gambari et al., Experimentia 41:673, 1985). After 10minutes incubation in the dark, the percentages of benzidine positivecells (hemoglobin positive cells) were determined by light microscopyusing an hemocytometer. Approximately 500 cells were counted for thedetermination of the percentages of benzidine positive cells. Cell sizewas also determined by light microscopy. Hemin (20 μg) was added to K562cells for 72 hours as a control for hemoglobin synthesis. Hemin wasobtained from Sigma-Aldrich Canada. TABLE 1 Evaluation of hemoglobinsynthesis (benzidine-positivity) and cell size of K562 cells SEQUENCE %of benzidine-positive cells Cell size None 5.4 normal SEQ ID NO: 1 17.4increased SEQ ID NO: 2 35.8 increased SEQ ID NO: 3 11.4 increased SEQ IDNO: 4 10.1 increased Hemin 17.8 normalAs shown in Table 1, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ IDNO: 4 induce the synthesis of hemoglobin by K562 cells and an increasein their cell size, two measures of erythroid differentiation.

EXAMPLE 4

Upregulation of Rh D in K562 cells cultured with SEQ ID NO. 2 or SEQ IDNO: 3.

The Rh D antigen is the most important antigen of the Rh blood groupsystem. In humans, the Rh D antigen is expressed solely on erythrocytes(Cartron, Blood Rev. 6:199, 1994). K562 cells were seeded in 1.0 ml at2.0×10⁵ cells/ml in 6-well flat-bottomed tissue culture plates for 72hours with 2.5, 10.0, 25.0, 50.0 or 100.0 μg of SEQ ID NO: 2 or SEQ IDNO: 3. The expression of Rh D at the cell surface was monitored by flowcytometry. After incubation, K562 cells were washed twice bycentrifugation with PBS and labeled with phycoerythrin (PE)-conjugatedanti-Rh D monoclonal antibody (IBGRL research product, Bristol,Netherlands) for 30 min at 4° C. After washing twice with PBS-1% bovineserum albumin, cellular fluorescence was then determined. Flow cytometrywas carried out on a FACSCalibur cell sorter (Becton Dickinson, SanJose, Calif., USA) and analyzed using the program CELLQuest (BectonDickinson). The fold increase in Rh D level over control (0 μgoligonucleotide) was determined. Untreated K562 cells were essentiallynegative for this marker. TABLE 2 Fold increase in Rh D level overcontrol in treated K562 with SEQ ID NO: 2 and SEQ ID NO: 3 Concentration(μg/ml) SEQUENCE 2.5 10 25 50 100 SEQ ID NO: 2 1.7 x 2.5 x 4.6 x 9.8 x15.4 x SEQ ID NO: 3 2.0 x 2.2 x 2.9 x 6.5 x  4.7 xAs shown in Table 2, SEQ ID NO: 2 and SEQ ID NO: 3 induced theexpression of Rh D antigen at the cell surface of K562, a measure oferythroid differentiation.

EXAMPLE 5

Upregulation of CD41a Antigen in K562 Cells Cultured with SEQ ID NO. 2or SEQ ID NO. 3.

The CD41a antigen, also named GpIIb/IIIa, is expressed on platelets andmegakaryocytes (Gruel et al., Blood 68:488, 1986). K562 cells wereseeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-well flat-bottomed tissueculture plates for 72 hours with 2.5, 10.0, 25.0, 50.0 or 100.0 μg ofSEQ ID NO: 2 or SEQ ID NO: 3. The expression of CD41a at the cellsurface was monitored by flow cytometry. After incubation, K562 cellswere washed twice by centrifugation with PBS and labeled withphycoerythrin (PE)-conjugated anti-CD41a monoclonal antibody (BDPharmingen, Mississauga, Ontario, Canada) for 30 min at 4° C. Afterwashing twice with PBS-1% bovine serum albumin, cellular fluorescencewas then determined. Flow cytometry was carried out on a FACSCaliburcell sorter (Becton Dickinson) and analyzed using the program CELLQuest(Becton Dickinson). The fold increase in CD41a level over control (0 μgoligonucleotide) was determined. Untreated K562 cells were essentiallynegative for this marker. TABLE 3 Fold increase in CD41a level overcontrol in treated K562 cells with SEQ ID NO: 2 or SEQ ID NO: 3Concentration (μg/ml) SEQUENCE 2.5 10 25 50 100 SEQ ID NO: 2 3.0 x 3.9 x12.3 x 20.6 x 18.9 x SEQ ID NO: 3 3.2 x 3.7 x  8.3 x 13.4 x 11.7 xAs shown in Table 3, SEQ ID NO: 2 and SEQ ID NO: 3 induced theexpression of CD41a antigen, a measure of megakaryocyte differentiation,at the cell surface of K562 cells.

EXAMPLE 6

Upregulation of CD14 in K562 Cells Cultured with SEQ ID NO. 2 or SEQ IDNO. 3.

The CD14 antigen is expressed at high levels on monocytes. Additionally,CD14 is expressed on interfollicular macrophages, reticular dendriticcells and some Langherans cells (Wright et al., Science 249:1434, 1990).K562 cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-wellflat-bottomed tissue culture plates for 72 hours with 2.5, 10.0, 25.0,50.0 or 100.0 μg of SEQ ID NO: 2 or SEQ ID NO: 3. The expression of CD14at the cell surface was monitored by flow cytometry. After incubation,K562 cells were washed twice by centrifugation with PBS and labeled withfluorescein isothiocyanate (FITC)-conjugated anti-CD14 monoclonalantibody (BD Pharmingen) for 30 min at 4° C. After washing twice withPBS-1% bovine serum albumin, cellular fluorescence was then determined.Flow cytometry was carried out on a FACSCalibur cell sorter (BectonDickinson) and analyzed using the program CELLQuest (Becton Dickinson).The fold increase in CD14 level over control (0 μg oligonucleotide) wasdetermined. Untreated K562 cells were essentially negative for thismarker. TABLE 4 Fold increase in CD14 level over control in K562 cellstreated with SEQ ID NO: 2 or SEQ ID NO: 3 Concentration (μg/ml) SEQUENCE2.5 10 25 50 100 SEQ ID NO: 2 1.8 x 1.9 x 3.9 x 7.2 x 9.8 x SEQ ID NO: 31.9 x 2.8 x 3.8 x 7.2 x 3.7 xAs shown in Table 4, SEQ ID NO: 2 and SEQ ID NO: 3 induced theexpression of CD14 antigen, a measure of monocyte differentiation, atthe cell surface of K562 cells.

EXAMPLE 7

Induction of CD14⁺Rh D⁺, CD14⁺CD41a⁺ and Rh D⁺CD41a⁺ Phenotype in K562Cells Cultured with SEQ ID NO. 2.

K562 cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-wellflat-bottomed tissue culture plates for 72 hours with 2.5, 10.0, 25.0,50.0 or 100.0 μg of SEQ ID NO: 2. The expression of CD14, CD41a and Rh Dat the cell surface was monitored by two dimensional flow cytometry.After incubation, K562 cells were washed twice by centrifugation withPBS and labeled with FITC-conjugated anti-CD14, PE-conjugated anti-CD41aand/or PE-conjugated anti-Rh D monoclonal antibody (BD Pharmingen) for30 min at 4° C. After washing twice with PBS-1% bovine serum albumin,cellular fluorescence was determined. Flow cytometry was carried out ona FACSCalibur cell sorter (Becton Dickinson) and analyzed using theprogram CELLQuest (Becton Dickinson). The fold increase in CD14⁺Rh D⁺,CD14⁺CD41a⁺ and Rh D⁺CD41a⁺ level over control (0 μg oligonucleotide)was determined. Untreated K562 cells were essentially negative for thesemarkers. TABLE 5 Fold increase in CD14⁺Rh D⁺, CD14⁺CD41a⁺ and RhD⁺CD41a⁺ levels in K562 treated with SEQ ID NO: 2 Concentration (μg/ml)PHENOTYPE 2.5 10 25 50 100 CD14⁺Rh D⁺ 4.0 x 7.0 x 16.0 x 34.0 x 41.0 xCD14⁺CD41a⁺ 2.0 x 2.0 x  4.0 x 11.0 x 22.0 x Rh D⁺CD41a⁺ 20.0 x  22.0 x 45.0 x 87.0 x 100.7 x As shown in Table 5, SEQ ID NO: 2 induced the differentiation of K562cells into cells with heterogeneous phenotypes.

EXAMPLE 8

Induction of CD14⁺Rh D⁺, CD14⁺CD41a⁺ and Rh D⁺CD41a⁺ Phenotype in K562Cells Cultured with SEQ ID NO: 3.

K562 cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-wellflat-bottomed tissue culture plates for 72 hours with 2.5, 10.0, 25.0,50.0 or 100.0 μg of SEQ ID NO: 3. The expression of CD14, CD41a and Rh Dat the cell surface was monitored by two dimensional flow cytometry.After incubation, K562 cells were washed twice by centrifugation withPBS and labeled with FITC-conjugated anti-CD14, PE-conjugated anti-CD41aand/or PE-conjugated anti-Rh D monoclonal antibody (BD Pharmingen) for30 min at 4° C. After washing twice with PBS-1% bovine serum albumin,cellular fluorescence was then determined. Flow cytometry was carriedout on a FACSCalibur cell sorter (Becton Dickinson) and analyzed usingthe program CELLQuest (Becton Dickinson). The fold increase in CD14⁺RhD⁺, CD14⁺CD41a⁺ and Rh D⁺CD41a⁺ level over control (0 μgoligonucleotide) was determined. Untreated K562 cells were essentiallynegative for these markers. TABLE 6 Fold increase in CD14⁺Rh D⁺,CD14⁺CD41a⁺ and Rh D⁺CD41a⁺ levels in K562 treated with SEQ ID NO: 3Concentration (μg/ml) PHENOTYPE 2.5 14 25 50 100 CD14⁺Rh D⁺ 7.0 x 8.0 x9.0 x 11.0 x 10.0 x CD14⁺CD41a⁺ 2.0 x 3.0 x 4.0 x  4.0 x  3.0 x RhD⁺CD41a⁺ 6.0 x 4.0 x 7.0 x 29.0 x 31.0 xAs shown in Table 6, SEQ ID NO: 3 induced the differentiation of K562cells into cells with heterogeneous phenotypes.

EXAMPLE 9

Inhibition of K562 Cell Growth by SEQ ID NO. 2 and SEQ ID NO. 3.

Terminal differentiation of K562 cells has been reported to stop theircellular growth (Bianchi et al., Biochem. Pharmacol. 60:31, 2000). K562cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-well flat-bottomedtissue culture plates for 72 hours with 1.0, 10.0 or 100.0 μg of SEQ IDNO: 2 or SEQ ID NO: 3. Cells were counted after 24, 48 and 72 hours ofincubation using light microscopy and trypan blue dye. TABLE 7 Number ofK562 cells (×10⁵) after treatment with SEQ ID NO: 2 or SEQ ID NO: 3 SEQID NO: 2 SEQ ID NO: 3 No oligo- 1.0 10.0 100.0 1.0 10.0 100.0 Hoursnucleotide μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml 24 3.6 3.1 2.5 2.6 3.32.7 3.2 48 7.4 7.8 6.4 2.2 8.4 7.1 3.4 72 11.5 11.3 8.5 1.7 11.2 8.4 3.3As shown in Table 7, SEQ ID NO: 2 and SEQ ID NO: 3 inhibited thecellular growth of K562 cells in a dose-dependent manner. The trypanblue exclusion assay demonstrated that treatment of K562 cells with SEQID NO: 2 or SEQ ID NO: 3 was not cytotoxic since no trypan blue dye wasincorporated by K562 cells.

EXAMPLE 10

Differentiation of Human Committed Erythroid Precursor by SEQ ID NO. 2

Glycophorin A is the major glycoprotein of the human erythrocytemembrane. Maturation of committed human erythroid precursors ischaracterized by the expression of glycophorin A at the cell surface andby an increase in intracellular granulosity (Daniel and Greens, VoxSang. S2:149, 2000; Wheater et al., Functional Histology, a text andcolor atlas, 2^(nd) edition, Churchill Livingstone, U.K., 1987). Humancommitted erythroid precursors defined by the cell surface glycoproteinCD36 were isolated from expanded human cord blood CD34+ progenitors bypositive immunoselection of CD36+ cells (Clonetics, San Diego, Calif.,USA). Human committed erythroid cells were seeded in 1.0 ml at 1.5×10⁵cells/ml in 6-well flat-bottomed tissue culture plates for 96 hours with100.0 μg of SEQ ID NO: 2 or SEQ ID NO: 3. The expression of glycophorinA at the cell surface and the intracellular granulosity were monitoredby flow cytometry. After 48 and 96 hours of incubation, human committederythroid cells were washed twice by centrifugation with PBS and labeledwith PE-conjugated glycophorin A monoclonal antibody (CaltagLaboratories, Burlingame, Calif., USA) for 30 min at 4° C. After washingtwice with PBS-1% bovine serum albumin, cellular fluorescence wasdetermined. The intracellular granulosity was determined by the measureof side light scatter (SSC) using a flow cytometer. Flow cytometry wascarried out on a FACSCalibur (Becton Dickinson) and analyzed using theprogram CELLQuest (Becton Dickinson). The percentages of cells inSSC^(hi) glycophorin A⁺ and in SSC^(lo) glycophorin A⁺ were determined.SSC^(hi) is defined as >450 units; SSC^(lo) is defined as <450 units.TABLE 8 Percentages of human committed erythroid precursor cells inSSC^(hi) glycophorin A⁺ and in SSC^(lo) glycophorin A⁺ after treatmentwith SEQ ID NO: 2 or SEQ ID NO: 3 SEQ ID SEQ ID None NO: 2 NO: 3 48 h 96h 48 h 96 h 48 h 96 h SSC^(hi)glycophorinA⁺ 0.9 1.2 8.0 12.8 0.6 0.4SSC^(lo)glycophorinA⁺ 9.1 3.8 9.3 18.1 7.4 2.2As shown in Table 8, SEQ ID NO: 2 induced the differentiation of humancommitted erythroid precursor cells in SSC^(hi) glycophorin A⁺ and inSSC^(lo) glycophorin A⁺ cells.

EXAMPLE 11

Effect of SEQ ID NO. 2 on Human Disseminated Chronic Myeloid LeukemiaK562 Cells in Severe Combined Immunodeficiency Mice

Forty female severe combined immunodeficiency mice (SCID mice) wereexposed to 1.8 Gy of radiation (rate: 7.5 Gy/h) from a γ source.Twenty-four hours after whole body irradiation (day 0), the 40 femaleSCID mice were weighed and randomized to form 4 groups (10 mice/group).The mean body weight of each group was not statistically different fromthe others (analysis of variance). Mice were injected intraperitoneally(ip) with 2.0×10⁷ K562 cells in 0.5 ml of RPMI-1640 medium. SEQ ID NO: 2(5′GGGTGG3′) resuspended in 0.9% sodium chloride USP was administratedip at 0.01, 0.1 and 1 mg per mouse per day from day 1 to day 29 (30days). A vehicle group was ip injected with vehicle (0.9% sodiumchloride USP) following the same schedule. The treatment schedule issummarized in the table below: TABLE 9 Dose/inj. (mg/ Vol./ Mice/ Routemouse/ inj. Treatment Group Treatment group Admin. inj.) (ml) schedule 1Vehicle 10 ip 0 0.250 Q1DX30 2 SEQ ID NO: 2 10 ip 0.01 0.250 Q1DX30 3SEQ ID NO: 2 10 ip 0.1 0.250 Q1DX30 4 SEQ ID NO: 2 10 ip 1 0.250 Q1DX30The experiment was stopped at 120 days when mice were sacrificed.Survival was recorded two times per week.

The test evaluation expressed as a percentage (T/C %) and as theincreased life span value (ILS %) of the control evaluation wasdetermined. These are measures of the effectiveness of the compoundstested. Survival systems indicate a degree of success when T/Cpercentages exceed 125 and ILS percentages exceed 25. T is the mediansurvival times of animals treated with drugs and C is the mediansurvival time of control animals. T/C % and ILS % is expressed asfollowing:ILS %=[(T−C)/C]×100T/C %=[T/C]×100

Statistical analysis was performed using StatView® (Abacus Concept,Berkeley, USA). Statistical analysis of the efficiency of the treatmentwas performed using the Bonferroni/Dunn test (ANOVA comparison). TABLE10 Survival time of SCID mice having human disseminated chronic myeloidK562 leukemia treated with SEQ ID NO: 2 TREATMENT Group 4: 1 mg Group 2:Group 3: SEQ ID Group 1: 0.01 mg 0.1 mg NO: 2 vehicle SEQ ID NO: 2 SEQID NO: 2 Survival Survival Survival time Survival time time time (days)(days) (days) (days) 38 120 85 49 54 57 34 120 46 54 57 41 54 75 75 5734 120 34 57 38 120 31 99 34 120 64 61 54 61 54 61 75 61 64 46 54 34 4146 34 38 34 49 Mean ± sd 48.6 ± 12.2 78.2 ± 34.9 52.1 ± 18.7 62.4 ± 24.6median 46 61 41 49 ILS % — 32.6 −12.2 6.5 T/C % — 132.6 89.1 106.5

As shown in Table 10, SEQ ID NO: 2, at 0.01 mg/mouse/day, significantlyincreased the life span of SCID mice having human disseminated chronicmyeloid K562 leukemia (p<0.05). After 120 days, 4 of 11 mice treatedwith SEQ ID NO:2 at 0.01 mg/mouse/day were alive while none of the 11untreated mice was alive.

EXAMPLE 12

Effect of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO. 3 and SEQ ID NO: 4 onDifferentiation of Bone Marrow-Derived Cells from Mice

The C57BL/6 mice are divided into 5 groups of 10 mice. Mice receivegamma-irradiation to induce a reduction in the number of bone marrowderived-cells and bone marrow precursor cells. On day 0, group 1 micereceive saline, group 2 receive SEQ ID NO: 1, group 3 receive SEQ ID NO:2, group 4 receive SEQ ID NO: 3, group 5 receive SEQ ID NO: 4. Sequencesresuspended in 0.9% sodium chloride USP are administrated ip at 0.01 mgper mouse per day for 7 days.

After 7 days of treatment, the mice are sacrificed. Cells present inperipheral blood and in bone marrow are counted and their phenotypedetermined by flow cytometry. Hemoglobin levels are also determined.Mice in groups 2, 3, 4 and 5 have more bone marrow-derived cells thanthe mice in group 1. Mice in groups 2, 3, 4 and 5 have more mature bonemarrow-derived cells than the mice in group 1. The levels of hemoglobinare more elevated in mice in groups 2, 3, 4 than in mice in group 1.

EXAMPLE 13

Effect of SEQ ID NO: 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 onCML in SCID Mice

K562 cells (2×10⁷ cells) are inoculated into SCID mice (severe combinedimmunodeficiency mice) as previously described (Beran et al., Hematol.Pathol. 8:135, 1994). The mice are divided into 5 groups of 10 mice. Onday 0, group 1 mice receive saline, group 2 mice receive SEQ ID NO: 1,group 3 mice receive SEQ ID NO: 2, group 4 receive SEQ ID NO: 3, group 5mice receive SEQ ID NO: 4. Sequences resuspended in 0.9% sodium chlorideUSP are administrated ip at 0.01 mg per mouse per day for 30 days.

After 30 days, the mice are sacrificed. Leukemic dissemination, leukemiccell phenotype and hemoglobin levels are analyzed. Mice in Group 1 havethe most leukemia cells and dissemination. Mice in groups 2, 3, 4 and 5have less leukemia cells and dissemination. Mice in groups 2, 3, 4 and 5show a higher number of differentiated K562 cells than mice in group 1.Mice in groups 2, 3, 4 and 5 show more hemoglobin synthesis than themice in group 1.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

1. A composition comprising a 3′-OH, 5′-OH, chemically unmodified, synthetic phosphodiester nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and a pharmaceutically acceptable carrier, wherein the composition is effective to induce differentiation of cells, to increase cells derived from pluripotent cells, or to treat disease associated with insufficient differentiation of cells when administered to the cells in vivo or in vitro.
 2. The composition of claim 1, wherein the induction of differentiation of cells is induction of differentiation of leukemia cells.
 3. The composition of claim 1, wherein the induction of differentiation of cells is induction of differentiation of pluripotent stem cells, myeloid stem cells, lymphoid stem cells, progenitor cells, immune cell precursors, or cells derived from the pluripotent stem cells, the myeloid stem cells, the lymphoid stem cells, the progenitor cells, or the immune cell precursors.
 4. The composition of claim 1, wherein the disease is leukemia, lymphoma, a non-malignant blood disorder, hemoglobinopathy, sickle cell disease, myelodysplastic syndrome, pancytopenia, anemia, thrombocytopenia or leukopenia.
 5. A method comprising administration of an amount of a composition comprising a 3′-OH, 5′-OH, chemically unmodified, synthetic phosphodiester nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and a pharmaceutically acceptable carrier, to an animal or a human wherein the amount is effective to induce differentiation of cells in the animal or the human.
 6. The method of claim 5, wherein the animal or the human has a disease associated with insufficient differentiation of cells.
 7. The method of claim 6, wherein the disease is leukemia, lymphoma, a non-malignant blood disorder, hemoglobinopathy, sickle cell disease, myelodysplastic syndrome, pancytopenia, anemia, thrombocytopenia or leukopenia.
 8. The method of claim 6, wherein the disease is leukemia.
 9. The method of claim 5, wherein induction of differentiation of cells is induction of erythrocyte-like phenotype, monocyte-like phenotype, megakaryocyte-like phenotype, inhibition of proliferation or induction of hemoglobin synthesis in cells.
 10. A method comprising administration of an amount of a composition comprising a 3′-OH, 5′-OH, chemically unmodified, synthetic phosphodiester nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and a pharmaceutically acceptable carrier, to an animal or a human, wherein the amount is effective to increase differentiation of pluripotent cells in the animal or the human.
 11. The method of claim 10, wherein the animal or the human has received chemotherapy or radiotherapy.
 12. The method of claim 4, further comprising administration of a therapeutic agent.
 13. The method of claim 12, wherein the therapeutic agent is a chemotherapeutic drug, an immunosuppressive agent, a differentiating agent, an immunotherapeutic agent, an antimicrobial agent, an antiviral agent, radiotherapy, or a combination thereof.
 14. The method of claim 10, wherein the pluripotent cells are derived from bone marrow, liver, spleen, lymph nodes, thymus or cord blood.
 15. The method of claim 10, wherein the pluripotent cells are derived from bone marrow.
 16. A method comprising administration of an amount of a composition comprising a 3′-OH, 5′-OH, chemically unmodified, synthetic phosphodiester nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and a pharmaceutically acceptable carrier, to an animal or a human having a disease associated with insufficient differentiation of cells, wherein the amount is effective to induce differentiation of cells in the animal or the human.
 17. The method of claim 16, wherein the disease is leukemia and the amount is effective to treat the leukemia. 